Ca2+ dependent arrhythmias underlie or contribute to sudden cardiac death (SCO). Project 3 of the Marks Program Project Grant application seeks to investigate the subcellular basis of such arrhythmogenesis. Experiments are proposed to characterize quantitatively the cellular, subcellular, and molecular triggers that initiate fatal cardiac arrhythmias. Specifically Ca2* sparks and other cellular and subcellular Ca2+ signals will be measured in mouse models of Ca2+ dependent arrhythmogenesis. Preliminary data from the PI and other members of the PPG team suggests that increased sensitivity of the cardiac ryanodine receptor (RyR2), alterations in SR Ca2+ content, and changes in RyR2 macromolecular complex can each contribute to this process. Our approach enables us to investigate the central hypothesis of the PPG: SCO can be caused by disruption of molecular complexes and processes that, in normal hearts, underlie balanced regulation of cellular activity. Using patch clamp methods with confocal Ca2+ imaging at high temporal and spatial resolution, Ca2+ sparks and Ca2+ signals will be measured in single mouse ventricular myocytes. In some experiments intracellular sodium concentration, [Na+]i, will be measured. Additionally, step changes in intracellular Ca2+ will be imposed using a new flash photolysis method with improved resolution, sensitivity, and control. Mouse models will be used to investigate two important interrelated questions. (1) How does modulation of the cardiac ryanodine receptor (RyR2) account for significant changes in cardiac Ca2+ signaling? (2) Can the properties of sarcolemmal ion channels contribute to pathological remodeling of cardiac Ca2+ signaling to produce a significant proarrhythmic substrate? Preliminary experiments from each of the three tightly linked projects of this PPG suggest that the planned experiments are likely to reveal novel regulatory elements in the control of subcellular Ca2+ signaling. The planned work should lay the foundation for improved understanding of Ca2+ signaling in SCO and provide the insight needed to devise novel molecular-targeted therapeutic strategies for treatment.